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First published online February 21, 2008
Stem Cells Vol. 26 No. 5 May 2008, pp. 1265 -1274
doi:10.1634/stemcells.2007-1067; www.StemCells.com
© 2008 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Hes1 Regulates Corneal Development and the Function of Corneal Epithelial Stem/Progenitor Cells

Takahiro Nakamuraa,b,c, Toshiyuki Ohtsukad, Eiichi Sekiyamaa, Leanne J. Coopere, Hiroshi Kokubud, Nigel J. Fullwoode, Yann Barrandonc, Ryoichiro Kageyamad, Shigeru Kinoshitaa

aDepartment of Ophthalmology, Kyoto Prefectural University of Medicine, Graduate School of Medicine, Kyoto, Japan;
bResearch Center for Regenerative Medicine, Doshisha University, Kyoto, Japan;
cLaboratory of Stem Cell Dynamics, Ecole Polytechnique Federale de Lausanne and Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland;
dInstitute for Virus Research, Kyoto University, Kyoto, Japan;
eBiomedical Sciences, Lancaster University, Lancaster, United Kingdom

Key Words. Cornea • Development • Hes • Notch • Stem cell

Correspondence: Correspondence: Takahiro Nakamura M.D., Ph.D., Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602-0841 Japan. Telephone: 81-75-251-5578; Fax: 81-75-251-5663; e-mail: tnakamur{at}ophth.kpu-m.ac.jp

Received on December 14, 2007; accepted for publication on February 12, 2008.

First published online in STEM CELLS EXPRESS  February 21, 2008.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Hes1, a major target gene in Notch signaling, regulates the fate and differentiation of various cell types in many developmental systems. To gain a novel insight into the role of Hes1 in corneal tissue, we performed gain-of-function and loss-of-function studies. We show that corneal development was severely disturbed in Hes1-null mice. Hes1-null corneas manifested abnormal junctional specialization, cell differentiation, and less cell proliferation ability. Worthy of note, Hes1 is expressed mainly in the corneal epithelial stem/progenitor cells and is not detected in the differentiated corneal epithelial cells. Expression of Hes1 is closely linked with corneal epithelial stem/progenitor cell proliferation activity in vivo. Moreover, forced Hes1 expression inhibits the differentiation of corneal epithelial stem/progenitor cells and maintains these cells' undifferentiated state. Our data provide the first evidence that Hes1 regulates corneal development and the homeostatic function of corneal epithelial stem/progenitor cells.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
In most vertebrates, including humans, the majority of external information is gained through sight, one of the five senses, and the cornea is a very important avascular tissue related to the maintenance of this visual system. The cornea consists of a stratified, nonkeratinized surface epithelial cell layer; a thick, highly aligned collagenous stroma sparsely populated by corneal keratocytes; and an inner, single-cell-layered endothelium. Through the combination of these three cell layers, corneal tissue is kept optically clear, and ocular homeostasis and integrity are maintained. If any one of the three cell layers becomes damaged, the cornea cannot maintain its transparency, and visual acuity is severely disturbed. Thus, there exist many patients with various severe corneal diseases all over the world (135 million individuals) [1].

From the aspect of corneal regenerative therapy, many scientists have been investigating corneal development, morphogenesis, and the molecular mechanism of cornea-specific stem cells to better understand the role and function of the cornea, as well as to elucidate the most effective means for corneal tissue reconstruction. Embryonically, eyes of vertebrates develop from different tissue components, including surface ectoderm, optic vesicle, a lateral evagination from the wall of diencephalons, and surrounding mesenchyme. A region of the surface ectoderm defined by the lens placode gives rise to the corneal epithelium, as well as to the lens, whereas the corneal stroma and endothelium develop from cells originating in the neural crest. Each corneal cell is distinguished by its position, characteristic morphology, and expression profiles of various biochemical markers [2]; however, the key factors that regulate corneal development and morphogenesis have not been fully elucidated.

In general, stem cells (SCs) facilitate the maintenance of self-renewing tissues and organs [3–9]. SCs give rise to progeny (transient amplifying cells [TACs]) that have limited renewal capacity. Upon exhaustion of their proliferative potential, the rapidly proliferating TACs undergo terminal differentiation. With regard to corneal tissue, both the corneal stromal and endothelial SCs have yet to be exactly identified, and their exact locations are also not fully understood. In contrast, various studies indicate that corneal epithelial SCs reside in the basal layer of peripheral cornea in the limbal zone [10, 11], which is located between the cornea and conjunctiva. The corneal epithelial stem cell system is one of the most clearly defined and best understood systems in the body and is therefore an ideal model in which to investigate the role of regulatory molecules in stem cells [4, 12]. However, the molecular mechanism behind the function and regulation of corneal epithelial SCs is still not well understood.

Particular attention has recently focused on the Notch signaling, which is an evolutionarily conserved mechanism that regulates cell fate determination and differentiation during development of various types of tissues and organs [13, 14]. Activation of Notch receptors leads to the release of the intracellular domain and activates expression of the negative basic helix-loop-helix (bHLH) Hes genes, including Hes1 [15, 16]. Thus, Hes1 is one of several related proteins known to be induced directly by Notch signaling [17, 18]. Hes1 is reportedly expressed in various types of tissue and organs and generally maintains cells in an undifferentiated precursor state by negatively regulating bHLH transcription factors [19].

We previously reported that Hes1 regulates differentiation and morphogenesis of retinal neurons [20, 21]. Hes1 is expressed in retinal progenitor cells, and its expression decreases as differentiation proceeds. From the experimental results of retrovirus infection with Hes1, persistent expression of Hes1 blocks retinal development. In contrast, in the retina of Hes1-null mutant mice, differentiation was accelerated. Moreover, Hes1 operates as a general negative regulator of endodermal endocrine development and functions as a commitment switch in the epidermal lineage while also maintaining the neural-crest-derived melanocyte stem cells [22–25]. Thus, Hes1 has various important roles in the development of ectoderm, endoderm, and neural-crest-derived tissues and organs. However, as far as we know, there have been no detailed reports regarding the exact function of Hes1 in corneal tissue.

In this study, to demonstrate the function and role of Hes1 in the cornea, we performed gain-of-function and loss-of-function studies. In addition, we demonstrate the critical role of Hes1 in corneal epithelial stem/progenitor cell behavior. Thus, our data provide new insights into the underlying homeostatic regulation of corneal epithelial stem/progenitor cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Animals
Hes1 mutant fetuses were obtained by mating heterozygous males and females for Hes1-targeted mutation [26]. Corneal tissues were collected from embryonic day 13.5 (E13.5) to E18.5 and immediately after birth (postnatal day 0 [P0]), because Hes1-null mice die during gestation or within 1 day after birth. Postnatal wild-type corneas were also collected from P7 to P56. Genotyping of the embryos was performed by polymerase chain reaction with tail DNA. pHes-enhanced green fluorescent protein (EGFP) transgenic mice, in which green fluorescent protein (GFP) expression mimics endogenous Hes1 promoter activation, were generated using the previously reported methods [27]. All animals used for this study were maintained and handled according to the protocols approved by the Kyoto Prefectural University of Medicine.

Light and Electron Microscopy
Whole eyes were fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer for 2 hours. They were then washed three times in 0.1 M cacodylate buffer for 15 minutes and then postfixed in 2% osmium tetroxide for an additional 2 hours. They were washed again in cacodylate buffer before being passed through a graded alcohol series and embedded in Araldite resin (Agar Scientific, Stansted, U.K., http://www.agarscientific.com). Semithin sections (1 µm thick) were cut on an ultramicrotome (Ultracut E; Reichert-Jung, Vienna, Austria, http://www.leica-microsystems.com), counterstained with toluidine blue for 1 minute prior to examination on a light microscope (Reichert-Jung). Ultrathin sections were collected on naked copper grids and counterstained for 1 hour each with aqueous uranyl acetate and 2% phosphotungstic acid and 20 minutes with Reynold's lead citrate before being examined on a transmission electron microscope (JEM 1010; JEOL Ltd., Tokyo, http://www.jeol.com).

Antibodies
The following mouse monoclonal antibodies (mAbs) were used: anti-desmoplakin (1x dilution) (Progen, Heidelberg, Germany, http://www.progen.de), anti-integrin {alpha}6 (200x)/β1 (500x) (Chemicon, Temecula, CA, http://www.chemicon.com), anti-laminin5 (100x) (Chemicon), anti-p63 (100x) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), anti-keratin 3 (50x) (Progen)/19 (100x) (Novocastra Ltd., Newcastle upon Tyne, U.K., http://www.novocastra.co.uk), anti-ki67 (200x) (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml), and anti-cyclinD1 (100x) (Santa Cruz Biotechnology). The following rat mAb was used: anti-5-bromo-2'-deoxyuridine (BrdU) (200x) (Abcam, Cambridge, U.K., http://www.abcam.com). The following rabbit polyclonal antibodies (pAbs) were also used: anti-ZO1 (25x) (Zymed, South San Francisco, http://www.invitrogen.com/content.cfm?pageid=11356), anti-Hes1 (1,000x) (a kind gift from Dr. T. Sudo, Toray Industries, Japan), anti-keratin12 (200x) (Transgenic, Kumamoto, Japan, http://www.transgenic.co.jp/index.html), and anti-GFP (200x) (BD Pharmingen). The following goat pAb was also used: anti-keratin12 (100x) (Santa Cruz Biotechnology). Secondary antibodies were Alexa Fluor-488/594 anti-mouse IgG (1,500x), Alexa Fluor-488/594 anti-rabbit IgG (1,500x), Alexa Fluor-488 anti-goat IgG (1,500x) (Molecular Probes, Eugene, OR, http://probes.invitrogen.com), Cy3-conjugated anti-rat IgG (1,500x), and Cy2-conjugated anti-rabbit IgG (200x) (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com).

Immunohistochemistry
Immunohistochemical studies followed our previously described method [28]. Briefly, 7-µm-thick cryostat sections were obtained from unfixed tissue embedded in Tissue-Tek OCT compound (Miles Laboratories, Inc., Elkhart, IN, http://www.bayer.com/en/Homepage.aspx). After fixation with cold acetone for 10 minutes, the tissues were incubated with 2% bovine serum albumin and goat or donkey serum (room temperature [RT], 30 minutes). For GFP staining, samples were fixed with 4% paraformaldehyde, dehydrated in 25% sucrose, embedded in OCT compound, and sectioned. We further incubated with MOM Mouse IgG blocking reagent (Vectashield; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) when we used mouse mAbs (RT, 1 hour). The sections were then incubated (RT, 1 hour) with the appropriate primary antibody (simple antibody or a mixture of antibodies for double staining) and washed (three times) in phosphate-buffered saline (PBS) containing 0.15% Triton X-100 for 15 minutes. Control incubations were with the appropriate normal mouse, rat, rabbit, and goat IgG (Dako, Glostrup, Denmark, http://www.dako.com) at the same concentration as the primary antibody; the primary antibody for the respective specimen was omitted. After staining with the primary antibody, the sections were incubated with the appropriate secondary antibodies (RT, 1 hour), washed several times with PBS, coverslipped using antifading mounting medium containing propidium iodide or 4,6-diamidino-2-phenylindole (Vectashield), and examined under a confocal microscope (Olympus Fluoview; Olympus, Tokyo, http://www.olympus-global.com).

BrdU Labeling
According to the manufacturer's protocol (Zymed), we injected the pregnant mother mice (n = 3) with BrdU labeling reagent (1 ml/100 g) at the time of E18.5. After 2 hours, the mice were sacrificed, and the eye was then embedded in Tissue-Tek II OCT compound (Miles Laboratories).

To detect the label-retaining cell (LRC), two mini-osmotic pumps (model 2001; Alzet Osmotic Pumps, Cupertino, CA, http://www.alzet.com) were implanted into the subcutaneous back skin of Hes1 wild-type mice (6–8 weeks old; n = 5). Each animal received a total dose of approximately 960 µg of BrdU per day [29]. After continuous labeling for 14 days, pumps were surgically removed, and the mice were left untreated for up to 5 weeks and then sacrificed. Following this chase period, only slow-cycling cells retained their label and were considered LRCs.

Three-Dimensional Culture
To culture mouse corneal epithelial cells, we used our previously reported system [28, 30, 31]. Corneal epithelial explants including the corneal limbus (Hes1 wild-type and null; n = 6) were put onto denuded amniotic membrane substrate spread on the bottom of culture inserts and cocultured for 14 days with mitomycin C-inactivated 3T3 fibroblasts (2 x 104 cells per cm2). The culture medium consisted of a defined keratinocyte growth medium (ArBlast Co., Ltd., Kobe, Japan, http://www.arblast.jp) supplemented with 5% fetal bovine serum. Cultures were incubated at 37°C in a 5% CO2/95% air incubator; the medium was changed daily.

In Vivo Wound Model
Experimental mice (Hes1 wild-type mice, 8 weeks old; n = 12) were anesthetized by intraperitoneal injection of combined xylazine and ketamine. After administration of topical oxybuprocaine eye drops (Santen, Osaka, Japan, http://www.santen.co.jp/jp/index.jsp), central corneal epithelial debridement was performed using a no. 69 Beaver Blade under a stereomicroscope. After healing intervals of 12, 24, and 36 hours, the experimental mice were killed by overdose of pentobarbital sodium, and the eyes were enucleated and embedded in OCT compound. To observe the area of remaining epithelial defect at the different time points, green fluorescent staining was used.

Infection of Retrovirus
Retrovirus infection was performed as with our previously described method, which follows the National Institutes of Health guidelines [32]. For CLIG-Hes1, Hes1 cDNA was cloned into the EcoRI site of pCLIG. The retroviral DNAs were transfected into the ectopic packaging cells {Psi}2mp34, and 2 days later, the medium was recovered and concentrated as previously described [33]. Virus solution was applied to the mouse corneal epithelial equivalent up to 5 days and then fixed with 4% paraformaldehyde, dehydrated in 25% sucrose, embedded in OCT compound, and sectioned. Almost all of the cells infected with the CLIG-Hes1 virus successfully expressed both Hes1 and GFP.

Apoptosis Assay
The cells were directly stained by the In Situ Apoptosis Detection Kit (terminal deoxynucleotidyl transferase dUTP nick-end labeling [TUNEL] assay; Takara, Otsu, Japan, http://www.takara.co.jp).

Quantitative Evaluation
For statistical assessment of TUNEL-, BrdU-, and Ki67-labeled cells, 30 different sections from three independent mice (wild-type and null, respectively) were analyzed. For statistical assessment of retrovirus experiments, five different fields and six different sections were analyzed (total, 30 areas).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Expression Pattern of Hes1 in the Developing Cornea
We first examined the expression pattern of Hes1 in the developing cornea by immunohistochemical analysis. During the middle embryonic period (Hes1 wild-type, E13.5), Hes1 was moderately expressed in whole corneas, lenses, and retinas (Fig. 1A). At the late embryonic period (E18.5–P0), the expression of Hes1 was markedly reduced, and sporadic expression was observed only in the palpebral conjunctival epithelium (Fig. 1B). At P7, Hes1 expression was observed again in the palpebral conjunctival epithelium but not in the corneal limbal epithelium (Fig. 1C, 1D). At P14, prior to the eyelids being open, Hes1 expression was observed in the palpebral/bulbar conjunctival epithelium and was also occasionally observed in the corneal limbal epithelium (Fig. 1E, 1F). At P28 and P42, expression of Hes1 was clearly observed in the corneal limbal basal cells, which are generally believed to be putative corneal epithelial stem/progenitor cells, but it was not expressed in the differentiated corneal epithelial cells (Fig. 1G–1I). Thus, Hes1 expression clearly decreased during the embryonic period. In contrast, at postnatal periods, its expression again occurred mainly in the ocular surface epithelium, implying the possible link with the function of ocular surface epithelium.


Figure 1
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  		Figure 1. Expression pattern of Hes1 in the developing Co. (A): At the middle embryonic period (Hes1 wild-type; E13.5), Hes1 was moderately expressed in whole Co, Cj, Epi, l, and r. (B): At the late embryonic period (E18.5–P0), the expression of Hes1 was observed only in the palpebral conjunctival epithelium (arrows). (C, D): At P7, Hes1 expression was observed again in the palpebral conjunctival epithelium. (E, F): At P14, Hes1 expression was observed in the palpebral/bulbar conjunctival epithelium and was also occasionally observed in the corneal limbal epithelium. (G, H, I): At P28 and P42, expression of Hes1 was clearly observed in the corneal limbal basal cells. Dotted lines indicate the corneal limbal areas. Scale bars = 100 µm. Abbreviations: Cj, conjunctivas; Co, corneas; E, embryonic day; Epi, epidermis; l, lenses; P, postnatal day; PI, propidium iodide; r, retinas.

 
Corneal Development and Morphogenesis Was Severely Disturbed in Hes1-Null Mice
To investigate the roles of Hes1 in corneal development and morphogenesis, we attempted a loss-of-function analysis by examining the cornea of Hes1-null mice. A total of 334 eyes (108 Hes1[+/+], 174 Hes1[+/–], and 52 Hes1[–/–]) were examined. The cornea of wild-type eyes was clear and transparent in appearance. In contrast, the Hes1-null cornea was hazy, and abnormal vascularization was sometimes observed (Fig. 2A, 2B). Examination of wild-type eyes by conventional light microscopy showed a normal, well-organized eye that appeared healthy, with a well-developed cornea, lens, and retina (Fig. 2C). Hes1-null eyes were significantly smaller in size compared with the control wild-types (Fig. 2D). They also showed significant disruption to the overall organization of the eye and a marked reduction in corneal thickness that was most apparent in the stromal region; the sclera was also much thinner (Fig. 2E, 2F).


Figure 2
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  		Figure 2. Phenotypical and morphological examination of Hes1 wild-type and null eyes. (A): The Co of wild-type eyes was clear and transparent in appearance. (B): Hes1-null Co was hazy, and abnormal vascularization was sometimes observed. (C, E): Conventional light microscopy showed a normal well-organized eye that appeared healthy, with a well-developed Co, l, and r in wild-type eyes. (D, F): Hes1-null eyes were significantly smaller in size compared with the control wild-types and showed significant disruption to the overall organization of the eye. Arrowheads indicate the abnormal bv. (G): Transmission electron microscopy showed a normal corneal e with 2–3 layers. The corneal s was well organized, and it consisted primarily of collagen fibrils and numerous keratocytes. (I): In the e, cells were joined together by numerous desmosomal junctions (arrowheads). (H): The s was significantly thinner than in normal wild-type Co. In some places, there was evidence of vascularization, with many bv. (J): The e was composed of only one cell layer, or at most two. (K): Quantitative examination showed a highly significant difference between total thicknesses of the Hes1-null Co compared with wild-types in central and Peri regions, respectively (n = 6). Arrows indicate the full thickness of the Co (E, F) and corneal e (I, J), respectively. Scale bars = 80 µm (C, D), 40 µm (E, F), 10 µm (G, H), and 1 µm (I, J). Abbreviations: bv, blood vessels; Co, cornea; e, epithelium; l, lens; P, postnatal day; Peri, peripheral; r, retina; s, stroma.

 
Examination of Hes1 wild-type corneas by transmission electron microscopy showed a normal corneal epithelium with 2–3 layers (Fig. 2G, 2I). Cells appeared well developed, with healthy cytoplasm and organelles, and were joined together by numerous desmosomal junctions (Fig. 2I). The corneal stroma was well organized, and it consisted primarily of collagen fibrils and numerous keratocytes, as would be expected in normal cornea. Examination of Hes1-null corneas showed considerable morphological differences compared with wild-types (Fig. 2H, 2J). The stroma was significantly thinner than in normal wild-type corneas, and in some places there was evidence of vascularization, with many blood vessels present underneath the stroma (Fig. 2H). In addition, the epithelium was composed of only one cell layer, or at most two (Fig. 2J). There appeared to be no evidence of normal stratification or differentiation, and the cells frequently appeared to be necrotic or apoptotic, with many vacuoles found in the cytoplasm and/or nucleus and also in organelles (Fig. 2J). Quantitative examination showed a highly significant difference between total thicknesses of the Hes1-null cornea compared with wild-type in the central and peripheral regions, respectively (Fig. 2K). These findings indicated that corneal development and morphogenesis was severely disturbed in Hes1-null mice, thus implying the critical role of Hes1 in embryonic corneal development.

Cell Biological Characteristics of the Hes1 Wild-Type and Hes1-Null Corneas
Using immunofluorescence and specific cell biological markers, we studied the cell biological characteristics of the Hes1 wild-type and null corneas (E17.5–P0). ZO-1, a tight-junction-related component, was expressed in the apical surface on Hes1 wild-type but not Hes1-null corneal epithelium (Fig. 3A, 3B). Desmoplakin, a cell-cell junction component, was clearly expressed in the Hes1 wild-type corneal epithelium; there was moderate desmoplakin expression on Hes1-null corneal epithelium (Fig. 3C, 3D). The basement-membrane assembly proteins integrin {alpha}6 and laminin5 clearly showed linear positive staining on the basement membrane side of Hes1 wild-type corneal epithelium, but in contrast, they showed faint or nonpositive staining in Hes1-null corneas (Fig. 3E–3H).


Figure 3
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  		Figure 3. Cell biological characteristics of Hes1 wild-type and null corneas. (A–N): Immunofluorescence of ZO-1 (A, B), desmoplakin (C, D), integrin {alpha}6 (E, F), laminin5 (G, H), integrin β1 (I, J), keratin 12 (K, L), and p63 (M, N) in Hes1 wild-type and null corneas, respectively. (O, P, S): The percentage of TUNEL(+) cells in Hes1-null corneas was significantly higher than that in the Hes1 wild-type corneas (arrows; *, p < .01; n = 3). (Q, R, T): The percentage of BrdU pulse labeling cells in Hes1 wild-type corneas was significantly higher than that in the Hes1-null corneas (*, p < .01; n = 3). Scale bars = 100 µm. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; E, embryonic day; P, postnatal day; TUNEL, TaT-mediated dUTP-biotin nick-end labeling.

 
Integrin β1, whose high expression is reportedly a marker of putative keratinocyte stem/progenitor cells, was expressed in nearly all epithelial cells on Hes1 wild-type corneas; no integrin β1 staining was observed in Hes1-null corneas (Fig. 3I, 3J). Keratin 12, a cornea-specific differentiation marker, was clearly expressed in the Hes1 wild-type corneas, whereas faint staining was observed in Hes1-null corneas (E17.5) (Fig. 3K, 3L). p63, which is strongly expressed in the basal layers or stem/progenitor cells of various epithelial tissues and regulates the proliferation and differentiation of those tissues [12, 34–37], is sporadically expressed in the basal cells of Hes1 wild-type corneal epithelium but not Hes1-null corneas (Fig. 3M, 3N).

In situ apoptosis cell detection experiments showed that the percentage of TaT-mediated duTP-biotin nick-end labeling (+) cells in Hes1-null corneas was significantly higher than that in the Hes1 wild-type corneas (Fig. 3O, 3P, 3S) (p < .01). Finally, cell proliferation ability was examined by incorporation of BrdU pulse labeling. The percentage of BrdU labeling cells in Hes1 wild-type corneas was significantly higher than that in the Hes1-null corneas (Fig. 3Q, 3R, 3T) (p < .01). These findings indicated that only Hes1 wild-type corneas manifested normal junctional specialization and normal cell differentiation and proliferation ability and that Hes1-null corneas manifested abnormal cell differentiation and less cell proliferation ability.

Three-Dimensional Corneal Epithelial Culture System
Since Hes1 is clearly expressed in the corneal limbal epithelial cells, we next examined the function of Hes1 in the corneal epithelium. Because all Hes1-null mice died either during gestation or within 1 day after birth, we were not able to examine the postnatal corneal epithelial development and function of Hes1-null mice. Our previous studies demonstrated that corneal limbal epithelial cells cultivated using appropriate growth factors and substrates have morphological and cell biological characteristics similar to those of in vivo normal corneal epithelium, thus indicating that these corneal epithelial equivalents closely mimic corneal epithelial development and morphogenesis in vivo [28, 30, 31]. Therefore, the problem of premature death of Hes1-null mice was overcome by studying the embryonic corneal preparations maintained in three-dimensional (3D) culture.

We cultivated corneal epithelium of Hes1 wild-type and null mice on an amniotic membrane substrate. After 14 days of cultivation, Hes1 wild-type cells formed 4–5 layers exhibiting well-conserved columnar basal cells and progressive flattening toward the surface, indicating that these corneal epithelial equivalents mimic the in vivo corneal epithelium (Fig. 4A). On the other hand, Hes1-null corneal cells grew in monolayers made up of elongated, differentiated cells (Fig. 4B). Keratin 12 was expressed in the Hes1 wild-type corneal equivalent, whereas faint staining was observed in the Hes1-null corneal equivalent (Fig. 4C, 4D). Keratin 19 and p63, putative corneal epithelial stem/progenitor cell-associated molecules [38], were expressed in the basal cells of the Hes1 wild-type equivalent, whereas no staining was observed in the Hes1-null equivalent (Fig. 4E–4H). To assess the cell-cycling status of these cultivated corneal epithelial equivalents, we examined their expression of Ki67. In both corneal epithelial equivalents, Ki67-labeled cells constituted 65.1% ± 3.17% and 14.3% ± 2.72% (SD), respectively, rendering the difference in the Ki67-labeling index statistically significant (*, p < .01; t test) (Fig. 4I, 4J, 4R). ZO-1 and integrin {alpha}6 were expressed in the apical surface and basement membrane side on the Hes1 wild-type and null equivalent, respectively (Fig. 4K–4N). Finally, we examined the expression of Hes1 in these cultivated corneal epithelial equivalents and found that Hes1 is sporadically expressed in the basal cells of wild-type equivalents but not in the Hes1-null equivalents (Fig. 4O, 4P). To further confirm these expression patterns of Hes1 in corneal epithelial equivalents, we used pHes1-d2EGFP (destabilized EGFP) mice, in which GFP expression mimics endogenous Hes1 promoter activation [27], and found that the expression pattern was similar to that of the Hes1 wild-type equivalent (Fig. 4Q). From these results, we demonstrated that the corneal epithelial phenotype of Hes1-null mice did not show the normal cell differentiation and proliferation activity and may lack the epithelial stem cell functions, indicating that the Hes1 gene severely affects the corneal epithelial function.


Figure 4
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  		Figure 4. Three-dimensional culture of corneal epithelial cells of Hes1 wild-type and null mice. (A, B): H&E staining of Hes1 wild-type and null corneal epithelial equivalents, respectively. (C–P): Immunofluorescence of K12 (C, D), K19 (E, F), p63 (G, H), Ki67 (I, J), ZO-1 (K, L), integrin {alpha}6 (M, N), and Hes1 (O, P) in Hes1 wild-type and null corneal epithelial equivalents, respectively. (R): In both corneal epithelial equivalents, Ki67-labeled cells constituted 65.1% ± 3.17% and 14.3% ± 2.72% (SD), respectively, rendering the difference in the Ki67-labeling index statistically significant (*, p < .01; t test; n = 3). (Q): pHes1–d2EGFP mouse corneal epithelial equivalent showed that the expression pattern was similar to that of the Hes1 wild-type equivalents. Scale bar = 50 µm. Abbreviation: EGFP, enhanced green fluorescent protein.

 
Cell Biological Relationship Between Hes1 and Epithelial Stem/Progenitor Cell-Related Molecules
To examine the relationship between Hes1 expression and proposed epithelial stem/progenitor cell-related molecules, we examined the double staining of Hes1 and proposed stem/progenitor cell-related molecules. In the adult mice (P56), Hes1 expression was clearly observed in the corneal limbal basal cells, but Hes1 was not expressed in the differentiated corneal epithelial cells (Fig. 5A). To further confirm the expression pattern of Hes1 in mouse corneal epithelial cells, we used pHes1-d2EGFP (destabilized EGFP) mice and found that the expression patterns were similar (Fig. 6B). Expression of keratins 3 and 12, the cornea-specific differentiation marker and the negative marker for corneal epithelial stem cells, respectively, was not colocalized with Hes1 (Fig. 5C, 5E). BrdU LRCs, putative corneal epithelial stem cells, were restrictedly observed in the corneal limbal epithelium and were also colocalized with Hes1 (Fig. 5D). Finally, Hes1 was invariably colocalized with p63 in the corneal limbal zone (Fig. 5F). These findings indicated that Hes1 has a strong relationship with proposed epithelial stem/progenitor cell-related molecules.


Figure 5
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  		Figure 5. Cell biological relationship between Hes1 and epithelial stem/progenitor cell-related molecules. (A): In the wild-type adult mice (postnatal day 56), Hes1 expression was observed in the corneal limbal basal cells (arrows). (B): pHes1–d2EGFP mouse cornea showed that the expression pattern was similar to that of the wild-type cornea. (C, E): Expression of keratins 3 and 12 was not colocalized with Hes1. (D): 5-Bromo-2'-deoxyuridine LRCs were restrictedly observed in the corneal limbal epithelium and were also colocalized with Hes1 (arrow). (F): Hes1 was invariably colocalized with p63 in the corneal limbal zone (arrows). Dotted lines indicate the corneal surface. Scale bars = 50 µm. Abbreviations: LRC, label-retaining cell; PI, propidium iodide.

 


Figure 6
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  		Figure 6. Physiological function of Hes1 in corneal wounding model. (A–D): Green fluorescent staining was used to identify the area of remaining epithelial defect at each time point (0, 12, 24, and 36 h; total n = 12). (E): Before debridement, the expression of Hes1 was restricted to the corneal limbal area. (F, G): After 12 h of debridement, the expression of Hes1 was seen in the area immediately adjacent to the limbal area ([G], arrows) and was not observed at the migrating edge (F). (H): The changes in the distribution of Hes1(+) cells were most evident at 24 h after wounding (arrows). (I, N): The expression of Hes1 was resituated in the limbal area at 36 h after wounding. (J): Before debridement, most of the Ki67(+) cells were observed in the peripheral and central corneal epithelium, but not in the limbal areas. (K, L): The number of Hes1(+)-Ki67(+) cells was gradually increased (arrows), and these cells were observed outside the limbal areas. (M, O): When the corneal defects were completely sealed, the percentage of Hes1(+)-Ki67(+) cells was gradually decreased. Asterisks indicate the border between corneal and limbal areas. Arrowheads indicate the iridocorneal angle. Dotted lines indicate the corneal surface. Scale bar = 100 µm. Abbreviation: h, hours.

 
Temporal Expression of Hes1 in Corneal Wound Healing
In the adult mice, the expression of Hes1 was restricted to the corneal limbal area, which is thought to be the location of corneal epithelial stem/progenitor cells, so we next examined the physiological function of Hes1 in corneal epithelial integrity using a corneal wound healing model. In our experiments, 2 mm (large wound) and less than 1 mm (small wound) central corneal debridement were resurfaced within 24–36 hours and 18 hours, respectively. Green fluorescent staining was used to identify the area of remaining epithelial defect at all time points (0, 12, 24, and 36 hours) (Fig. 6A–6D; supplemental online Fig. 1A–1D). Before large wounding (which means the physiological condition in vivo), the expression of Hes1 was restricted to the corneal limbal area (Fig. 6E). Twelve hours after large wounding, the expression of Hes1 was seen in the area immediately adjacent to the limbal epithelium but not in the central areas (Fig. 6F, 6G). Those changes in the distribution of Hes1(+) cells were most evident at 24 hours after wounding (Fig. 6H). Interestingly, the expression of Hes1 was resituated in the limbal area at 36 hours after wounding, at which point corneal epithelial defects had completely recovered (Fig. 6I, 6N). In contrast, small wounding could induce only a little change or no changes in the distribution of Hes1(+) cells (supplemental online Fig. 1E–1P), suggesting that activation of limbal epithelial function after wounding depends on the size of corneal epithelial damage, and change in the distribution of Hes1(+) cells after large wounding would indicate the centripetal movement of corneal limbal epithelium rather than the induction of the Hes1 expression.

To elucidate the relationship between the expression of Hes1 and cell proliferation activity, we next examined the double staining of Hes1 and Ki67, a marker for cell proliferation. Before large debridement, most of the Ki67(+) cells were observed in the peripheral and central corneal epithelium but not in the limbal areas (Fig. 6J). The number of Hes1(+)-Ki67(+) cells was gradually increased, and we could observe the changes in their distribution (Fig. 6K, 6L). When the corneal defects were completely sealed, the percentage of Hes1(+)-Ki67(+) cells was gradually decreased (Fig. 6M, 6O). These results indicated that in the in vivo situation, expression of Hes1 was closely linked with corneal limbal epithelial cell proliferation activity and Hes1(+) cells may act as a cell source for corneal epithelial regeneration.

Persistent Hes1 Expression Reduces Cell Proliferation and Regulates Undifferentiated Cells
Finally, we examined the effects of persistent Hes1 expression on corneal epithelial cells. Corneal epithelial equivalents (3D culture cells) were infected with the retrovirus CLIG, which directs EGFP expression [39], and CLIG-Hes1, which directs persistent Hes1 expression in addition to EGFP (Fig. 7A). Since the retrovirus is infectious only in dividing cells, we were able to monitor the fate of the virus-infected corneal epithelial cells. Cells infected with CLIG proliferated efficiently, and expression of GFP was strikingly increased at 120 hours after infection (Fig. 7B). In contrast, cells infected with CLIG-Hes1 proliferated less efficiently (Fig. 7B). The percentages of GFP-positive cells were 36.2% ± 7.33% (CLIG) and 15.7% ± 4.39% (CLIG-Hes1) at 120 hours after infection, respectively (p < .01) (Fig. 7C).


Figure 7
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  		Figure 7. Regulation of cell proliferation and differentiation by persistent Hes1 expression. (A): The schematic structures of recombinant retroviruses. (B): Cells infected with CLIG proliferated efficiently, and expression of GFP was strikingly increased at 120 h after infection. In contrast, cells infected with CLIG-Hes1 proliferated less efficiently. (C): The percentages of GFP-positive cells were 36.2% ± 7.33% (CLIG) and 15.7% ± 4.39% (CLIG-Hes1) at 120 h after infection, respectively (*, p < .01; n = 3). (D): The percentage of TUNEL-positive cells infected with CLIG was not different from that of those infected with CLIG-Hes1. (E): The ratios of cells positive for cyclin D1 were increased when CLIG-Hes1 was infected (*, p < .01; n = 3), although Ki67-positive cell ratios were not significantly changed. (F–H): In these series, there was a distinct tendency to increase the double-positive cells ratios of p63-GFP and K19-GFP (arrows) per total GFP-positive cells when infected with CLIG-Hes1 compared with CLIG (*, p < .01; n = 3). Scale bars = 20 µm. Abbreviations: %, percent; GFP, green fluorescent protein; h, hours; IRES, the internal ribosome entry site; LTR, long term repeat.

 
We also examined the TUNEL assay to demonstrate whether cell death is responsible for the reduction of cell proliferation. In our experiments, there was no tendency toward increased cell death when cells were infected with CLIG-Hes1 compared with CLIG, suggesting that persistent Hes1 expression reduces the cell proliferation rate without significant cell death (Fig. 7D). In agreement with these findings, ratios of cells positive for cyclin D1, a G1-specific marker, were increased when CLIG-Hes1 was infected (p < .01), although Ki67-positive cell ratios were not significantly changed (Fig. 7E). In view of these findings, it is likely that persistent Hes1 expression prolongs the G1 phase, thereby reducing cell proliferation.

To investigate the effects of persistent Hes1 expression on corneal epithelial cell differentiation, we performed double immunostaining for GFP and the putative corneal epithelial stem/progenitor cell-related molecules p63 and Keratin19. In these series, there was a distinct tendency toward increased double-positive cell ratios of p63-GFP and K19-GFP per total GFP-positive cells when infected with CLIG-Hes1 compared with CLIG, suggesting that forced Hes1 expression tends to inhibit the differentiation of corneal epithelial stem/progenitor cells and maintain these cells' undifferentiated state (Fig. 7F–7H).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Corneal tissue is extremely important since most mammals acquire the majority of their external information through it. In this study, we demonstrated that in the absence of the bHLH factor gene Hes1, corneal development and morphogenesis is severely disturbed and cornea tissue revealed abnormal cell differentiation and less proliferation ability. We also demonstrated that Hes1 was observed mainly in the ocular surface epithelium and has a strong relationship with the function of corneal epithelial stem/progenitor cells. Finally, persistent Hes1 expression reduced corneal epithelial cell proliferation and maintained these cells' undifferentiated state. These results provide the first evidence that Hes1 plays a critical role in the regulation of corneal epithelial integrity.

Abnormal cell differentiation occurred in the Hes1-null corneas. Although there existed some phenotypic variations in each examined sample, a decreased expression of K12 and a relatively increased level of TUNEL-positive cells were both observed in the Hes1-null corneas. Furthermore, K12 was still less expressed in the 3D corneal equivalents of Hes1-null mice. In contrast, it has been reported that Hes1-null mutation leads to premature neural differentiation and severe neural tube defects [25]. Unlike the neural tissues, however, the corneal phenotype of Hes1-null mice did not show the accelerated differentiation. In view of these results, we theorized that these abnormal differentiations could reflect the endogenous defects of the function of Hes1, rather than the secondary consequence of their accelerated differentiation and maturation.

We tried to examine the function of Hes1 in corneal epithelial stem/progenitor cells; however, the loss-of-function study of Hes1 was hampered by the embryonic lethality. Fortunately, we have established the 3D culture system of corneal epithelial SCs, which mimics the in vivo normal corneal epithelium. We therefore applied this system to Hes1-null corneas. We previously reported that Hes1 is involved in various aspects of cellular regulation, including SC maintenance [40]. Hes1-null corneal epithelial equivalents showed decreased or no expressions of corneal epithelial stem/progenitor cell-related molecules (K19 and p63) and a decreased level of cell proliferation activity (Ki67), suggesting that Hes1-null corneal epithelium may not possess the epithelial SC functions.

We thus demonstrated that corneal development and phenotype was severely disturbed in Hes1-null mice; however, we should interpret these findings carefully. Ubiquitous knockout of Hes1 includes gross abnormalities in growth and differentiation not only of the cornea but also of the lens and retina [20, 41]. This raises the question of whether the defect that is reported in this article might be secondary, even though Hes1 expression is unique in the corneal tissue. Therefore, further investigation using the conditional knockout model is needed to clarify these points.

It is now well documented that corneal epithelial SCs are not uniformly distributed throughout the entire corneal epithelial basal layer but are preferentially located in the corneal limbal basal layer. They produce undifferentiated progeny with limited proliferative potential (TAC) that migrate centripetally from the corneal limbus to replace or populate the cells under physiological conditions or at the time of wound stimuli. As far as we know, there have been few reports regarding corneal epithelial SC activation, function, and pattern of cell migration in vivo with links to putative SC regulatory factors. Interestingly, in the large wound experiments in our mice, centripetal movement of Hes1-positive cells was gradually observed and was most evident at 24 hours after wounding. It is worthy of note that movement naturally disappeared when the corneal epithelial wounding was completely sealed. Since Hes1 can be used to identify the corneal limbal phenotype, we presume that these centripetal movements suggest the migration of corneal epithelial progeny. Furthermore, disappearance of Hes1-positive cells outside the limbus at the time of wound sealing might suggest the differentiation process of corneal epithelium (ex. stem cells/early TAC to TAC). Thus, corneal large wounding stimuli cause the centripetal movement of Hes1-positive corneal limbal basal cells and the transient upregulation of Hes1, indicating that Hes1 plays an important role in corneal epithelial cell maintenance.

From the gain-of-function study using retrovirus infection, we demonstrated that a persistent and high level of Hes1 expression not only reduces cell proliferation activity but also tends to inhibit the differentiation of corneal epithelial stem/progenitor cells and maintain these cells' undifferentiated state. These findings are consistent with previous reports. In the central nervous system, persistent Hes1 expression inhibits both neuronal and glial differentiation and may keep the cells uncommitted to either neuronal or glial fates [42]. This effect is very similar to that of Notch signaling; activated Notch blocks cell fate commitment in both Drosophila and vertebrate [14], whereas Notch signaling has pleiotropic effects, such as binary cell fate decisions, induction of differentiation, and tumorigenesis [43–45]. In view of these, additional studies regarding the crosstalk between Notch and Hes are needed to understand their exact function in the corneal tissue.

In the corneal wound model, Hes1 expression correlates with the cell proliferation activity; however, overexpression of Hes1 in the 3D corneal culture model was shown to suppress cell proliferation. We think that the expression level of Hes1 in an in vivo corneal wound model is quite different from that in an in vitro retrovirus infection model. It has been reported that Hes1 mRNA is increased when Hes1 protein levels are low because Hes1 protein represses its own transcription by the negative feedback loop [46]. In the corneal wound model its expression level is under the endogenous range; therefore, cell proliferation activity is regulated by the Hes1 negative feedback loop. In contrast, the Hes1 expression level of the virus-infection model is substantially higher, and cells cannot be regulated by this negative feedback, with the result that persistent and high Hes1 expression reduces the cell proliferation. These findings are also found in the developing central nervous system [32].


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosure of Potential...
 Acknowledgments
 References
 
Our studies are the first demonstration of the function of the Notch-related gene Hes1 in the cornea. Many genes must regulate corneal development and morphogenesis, but it remains to be determined how their exact temporal expression is controlled. Furthermore, the function of corneal stem cells during embryonic and postnatal development is likely affected by the changes in the sets of various transcriptional factors expressed in corneal stem cells; however, it is not well known how these changes occur. Additional cell biological studies regarding the temporal changes in stem cell-regulatory gene expression are needed to understand the basic characteristics of corneal stem cells. In addition, greater knowledge regarding corneal stem cells will provide a foundation for the development of treatments for various corneal diseases.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Hisayo Saito and Tomoko Horikiri for assisting with the experimental procedures and John Bush for editing the manuscript. This study was supported in part by Grants-in-Aid for scientific research from the Japanese Ministry of Education, Culture, Sports, Science and Technology; the Swiss National Science Foundation (3100A0-104160); the Ecole Polytechnique Federale de Lausanne; the Centre Hospitalier Universitaire Vaudois; Euro Stem Cell; and Biotechnology and Biological Sciences Research Council in the United Kingdom.


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 

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